CN103596587A - Filovirus fusion proteins and their uses - Google Patents

Abstract

This invention provides fusion proteins comprising a Filovirus glycoprotein segment and an immunoglobulin polypeptide segment. The fusion proteins are useful in immunogenic compositions to protect against infections by Filoviruses, such as Ebola virus, in both humans and non-human animals. The fusion proteins are also useful in diagnostic assays to detect Filovirus infections.

Description

Filovirus fusion rotein and application thereof

Technical field

The present invention relates to the application of Filovirus (Filovirus) glycoprotein fusion rotein in prevention and the infection of diagnosis Filovirus.

Technical background

Ebola virus (EBOV) and Marburg virus (MARV) are the members of Filoviridae (Filoviridae), Filoviridae is the virus family that is categorized as " category-A " biological warfare agent, cause serious hemorrhagic fever in people and inhuman Primate, M & M is up to 90% (Sanchez etc., Filoviridae:Marburg and Ebola viruses(Filoviridae: Marburg and Ebola virus), 1409-1448 page, include in D.M.Knipe, P.M.Howley, D.E.Griffin, M.A.Martin, R.A.Lamb, B.Roizman and S.E.Straus (volume), Fields Virology(< < Fei Shi virusology > >), the 5th edition, the Donald Lippincott Williams of philadelphia, pa and Louis Wilkins publishing company (Lippincott Williams & Wilkins) 2007).After the short culture period of 4 – 10 days, there is suddenly the symptom identical with a lot of other viral infection in Filovirus infected individuals, comprises heating, feels cold, do not accommodate myalgia.MARV is stable and only has a kind in antigenicity, and EBOV is more variable and have five kinds.At the year ends 2007, in Uganda, Ben Dibujiao (Bundibugyo) EBOV (Towner etc., PLoS Pathog2008 November have been broken out recently; 4 (11): e1000212), compare the EBOV kind of Zaire (Zaire), the Sudan (Sudan) or Christopher Eccleston (Reston), itself and the Ivory Coast (Ivory Coast) plant more approaching.Zaire EBOV (ZEBOV) is associated with the highest fatality rate conventionally.The increase of EBOV outburst number in Africa outburst and in the recent period pig (Normile Science2009 January 23; 323 (5913): 451), caused that domestic animal can pass to lethal disease people's concern, this has given prominence to vaccine development and has comprised outburst in the urgency of interior quick diagnosis test.Vaccine based on Filovirus glycoprotein (GP), before clinical and in clinical evaluation, and does not currently have therapeutic agent to treat Filovirus to infect.Due to the license of safety and effective Filovirus vaccine can be needed to the several years, the individuality that diagnosis and isolation are infected is the main method of current restriction outburst.

Currently develop several Filovirus vaccine candidates, comprised recombinant adenovirus (Sullivan etc., PLoS Med2006 June of expressing EBOV GP; 3 (6): e177; Sullivan etc., Nature2003 August 7; 424 (6949): 681-4; With Sullivan etc., Nature2000 November 30; 408 (6812): 605-9), recombinant parainfluenza virus (Bukreyev etc., J Virol2007 June; 81 (12): 6379-88), restructuring Venezuelan equine encephalitis virus (Pushko etc., Vaccine2000 August 15; 19 (1): 142-53), recombinant replication type (Feldmann etc., PLoS Pathog2007 January; 3 (1): e2 and Jones etc., Nat Med2005 July; 11 (7): 786-90) and recombinant replication-defective type (Halfmann etc., J Virol2009 April; 83 (8): 3810-5) vesicular stomatitis virus and be loaded with virus-like particle (Warfield etc., the Proc Natl Acad Sci U S A2003 December 23 days of Filovirus GP; 100 (26): 15889-94 and Warfield etc., J Infect Dis2007 November 15; 196 supplementary issue 2:S430-7).That uses baculovirus expression Filovirus GP initially studies show that part protection, can work to the processing of glycosylation characteristic and GP in insect cell (Mellquist-Riemenschneider etc., Virus Res2003 April; 92 (2): 187-93).

Although make progress in this respect, the novel vaccine that also needs exploitation to infect for Filovirus.The invention solves these and other demand.

Summary of the invention

The invention provides the fusion rotein that comprises Filovirus glycoprotein fragment and immunoglobulin polypeptides fragment.In typical embodiment, described Filovirus glycoprotein fragment is for example from the ectodomain of Ebola virus (particularly Zaire Ebola virus Mayinga strain).The immunoglobulin heavy chain constant region polypeptide that described immunoglobulin polypeptides fragment can be IgG1 (being Fc fragment).In some embodiments, described fusion rotein also comprises the joint between Filovirus glycoprotein fragment and immunoglobulin polypeptides fragment.Exemplary fused albumen of the present invention is by there is no the SEQ ID NO:1 of joint and having the nucleic acid sequence encoding shown in the SEQ ID NO:3 of FLAG label joint.

The present invention also provides the immunogenic composition that comprises fusion rotein of the present invention.Described immunogenic composition can also comprise adjuvant.

The nucleic acid carrier that comprises fusion rotein nucleic acid sequence encoding of the present invention is also provided.Exemplary core acid sequence is shown in SEQ ID NO:1.

The present invention also provides the immunoreactive method of protection that induction is infected Filovirus in patient.Described method comprises the immunogenic composition of the present invention that gives patient's immunity effective dose.Described immunogenic composition can comprise fusion rotein of the present invention or its coding nucleic acid molecule.Described compositions can give with any number of ways.In some embodiments, described compositions intramuscular gives.

The present invention also provides the immunoreactive method of Filovirus in detection patient.Described method comprises makes patient's biological sample contact fusion rotein of the present invention, and detects body fluid or cell immune response.Can use ELISA, chemoluminescence test or fluorescent test to detect antibody.Cell immune response can detect by detecting IFN-γ, TNF-α or other cytokines and cell activation and proliferation test.

Another aspect of the present invention is the method that detects anti-Filovirus antibody (being neutralizing antibody) in patient's biological sample, and described patient uses Filovirus glycoprotein Fc fusion rotein or other immunogen immune.This method comprises makes biological sample contact express the recombinant vesicular stomatitis virus (VSV) (as Zaire Ebola virus Mayinga strain) of Filovirus GP, and evaluates residual infectivity in the cell that is subject to Filovirus infection.The described cell for this test can be Vero E6 cell.Or described restructuring VSV granule can be fixed on solid support, and anti-Filovirus antibody capable with standard method, for example ELISA, chemoluminescence test or fluorescent test detect.

Definition

Term " adjuvant " and " immunostimulant " be Alternate in this article, and is defined as the material that one or more cause that immune system stimulates.Herein, adjuvant is for strengthening the immunoreation to fusion rotein of the present invention.

Term used herein " amino terminal " (or " N-end ") and " carboxyl terminal " (or " C-end ") represent the particularly position in fusion rotein of the present invention of polypeptide.While allowing herein, the particular sequence or the fragment that relate to polypeptide or albumen are used these terms to represent near or phase loci.For example, some sequence that in polypeptide, relative canonical sequence is positioned at carboxyl terminal is positioned near the carboxyl terminal of canonical sequence, but must be at the carboxyl terminal of complete polypeptide.

When amino acid residue site for sequence, term " correspondence " refers to when the optimum comparison of sequence, the corresponding site of a plurality of sequences.

Term " expression vector " refers to the linear or circular DNA molecule that comprises polypeptide of interest encode fragment (as fusion rotein of the present invention), and optional being connected to of described polypeptide of interest encode fragment provides other fragments of transcribing.This other fragments comprise promoter and terminator sequence, and also can comprise one or more origin of replications, one or more selected marker, enhancer, polyadenylation signal etc.Expression vector is conventionally from antibacterial or viral DNA, and can comprise both elements.

When term under two or more nucleic acid or peptide sequence (as fusion rotein of the present invention and its coded polynucleotide) background " identical " or " homogeny " refer to compare and compare with regard to maximum correspondence, identical or have the same amino acid residue of specified percentage or two or more sequences or the subsequence of nucleotide, as use one of following sequence comparison algorithm or measure by visual inspection.

When the phrase " basic identical " under two nucleic acid of the present invention or polypeptide background refers to compare and compare with regard to maximum correspondence, two or more sequences have at least 60%, more preferably 65%, even more preferably 70%, still more preferably 75%, even more preferably 80% and most preferably 90-95% nucleotide or amino acid residue homogeny, as use one of following sequence comparison algorithm or measure by visual inspection.Preferably, on the basic identical sequence area being present at least about 50 residue length, more preferably on the region at least about 100 residues, and most preferably described sequence is basically identical at least 150 residues.In most preferred embodiments, described sequence is basically identical in the whole length of coding region.

For sequence comparison, generally a kind of sequence is used as to the canonical sequence with cycle tests comparison.While using sequence comparison algorithm, by cycle tests and reference sequence input computer, if desired specify subsequence coordinate, and specified sequence algorithm routine parameter.Then, sequence comparison algorithm calculates the sequence homogeny percent of the relative reference sequence of cycle tests according to the program parameter of appointment.

Can pass through, local homology's algorithm of Smith and Waterman for example, Adv.Appl.Math.2:482 (1981), by the sequence analysis algorithm of Needleman and Wunsch, J.Mol.Biol.48:443 (1970), by the similarity searching method of Pearson and Lipman, Proc.Nat ' l.Acad.Sci.USA85:2444 (1988), by computer, carry out these algorithms (GAP in Wisconsin hereditism's software kit of hereditism's calculating group (Genetics Computer Group) (Wisconsin Genetics Software Package), BESTFIT, FASTA and TFASTA, state of Wisconsin Madison (Madison, WI)) No. 575, science main road, or visual inspection (conventionally referring to, Current Protocols in Molecular Biology (the newly organized molecular biology experiment guide of < < > the >) (volume such as F.M.Ausubel, Current Protocols (the newly organized experiment guide > of < < >), Green publishes (the Greene Publishing Associates of affiliated company, Inc) and (the John Wiley & Sons of John Wei Lisen publishing company, Inc.) co-partnership company, (1995 supplementary issue) (Ausubel)) carry out optimal sequence comparison so that comparison.

The exemplary algorithm that is applicable to mensuration sequence homogeny percent and sequence similarity percent is BLAST and BLAST2.0 algorithm, is described in respectively (1977) Nucleic Acids Res.25:3389-3402 such as (1990) J.Mol.Biol.215:403-410 such as Altschul and Altschul.Carry out open obtain (the http://ncbi.nlm.nih.gov/) of software Ke Cong NCBI (National Center for Biotechnology Information) of BLAST analysis.This algorithm comprises: be first tested and appraised the short word that in search sequence, length is W and identify that high Grading sequence is to (HSP), when the word identical with length in database sequence compared they can mate or meet some on the occasion of threshold value scoring T.T is called adjacent words scoring threshold value (Altschul etc., the same).These initial adjacent words are hit as the seed that starts search, to find the longer HSP that contains them.Then, along each sequence, on both direction, extend this word and hit, until improve the comparison scoring of accumulation.With regard to nucleotide sequence, adopt parameter M (the award scoring of a pair of coupling residue; >0 always) and the N (point penalty of mispairing residue; <0 always) calculate accumulation scoring.With regard to aminoacid sequence, with rating matrix, calculate accumulation scoring.While there is following situation, end word and hit the extension in all directions: accumulation comparison scoring reduces X than its maximum acquisition value; Due to the accumulation of one or more negative scoring residue comparisons, below accumulation scoring vanishing or zero; Or reach the end of arbitrary sequence.BLAST algorithm parameter W, T and X determine sensitivity and the speed of comparison.The default value that BLASTN program (for nucleotide sequence) adopts is as follows: word length (W) 11, and expected value (E) 10, M=5, N=-4, and compare two chains.With regard to aminoacid sequence, BLASTP program is used default parameters, word length (W) 3, expected value (E) 10, use BLOSUM62 rating matrix (referring to Henikoff and Henikoff (1989) Proc Natl Acad Sci USA89:10915 (1989)).

Except sequence of calculation homogeny percent, BLAST algorithm also carries out the statistical analysis (referring to for example, Karlin and Altschul, Proc.Nat ' l.Acad.Sci.USA90:5873-5787 (1993)) of similarity between two sequences.A kind of similarity measurement that BLAST algorithm provides is minimum probability and (P (N)), and it shows to occur once in a while between two nucleotide or aminoacid sequence the probability of coupling.For example, if the minimum probability of test nucleic acid when with reference to nucleic acid comparison and be less than approximately 0.1 is more preferably less than approximately 0.01, be most preferably less than approximately 0.001, think that so this nucleic acid is similar to canonical sequence.

Illustrate that immunological cross-reaction can occur for polypeptide that two kinds of nucleotide sequences of the present invention or the essentially identical further index of polypeptide are the first nucleic acid codings and the polypeptide of the second nucleic acid coding, as described below.Therefore, for example,, when the difference of certain polypeptide and the second polypeptide is only for conservative replacement, these two kinds of peptides are conventionally basic identical.Two kinds of essentially identical another indexs of nucleotide sequence are the hybridization mutually under rigorous condition of two kinds of molecules, as described below.

" immunoglobulin " is as the serum albumin of antibody function in vertebrate organism body.Each immunoglobulin comprises light chain and heavy chain.Each chain comprises constant region and variable region.Have the heavy chain of 5 types, be expressed as α, δ, ε, γ and μ, it defines respectively Antibody types IgA, IgD, IgE, IgG and IgM.In people, IgG is comprised of four hypotypes that are called IgG1, IgG2, IgG3 and IgG4.The DNA sequence of encoding human and non-human immunoglobulin chain is well known.

Term " immunoglobulin heavy chain constant region polypeptide " represents wild type immunoglobulin heavy chain constant region or its variant.IgG constant region comprises C _h1, C _h2 and C _h3 domains and hinge region.

" Fc fragment " is the fragment of the CH corresponding with the immunoglobulin domain of Fc acceptor interaction.In IgG, IgA and IgD, the corresponding C in described Fc district _h2, and C _h3 domains and hinge region.At IgM and IgE, described Fc district comprises three CH (C _h2, C _h3 and C _h4).

" be operatively connected " and refer to that two or more DNA fragmentations combine, thereby with regard to its intended purpose synergism.For example, coded sequence is operatively connected promoter in correct frame, starts, and proceed to terminator by encode fragment thereby transcribe in promoter.

" polynucleotide " are conventionally from 5 ', to read to the deoxyribonucleotide of 3 ' end or list or the double-chain polymer of ribonucleotide base.Polynucleotide comprise RNA and DNA, and separable from natural origin, external synthetic or prepared by natural and synthetic molecular combinations.When described term is when the duplex molecule, it is for representing entire length, and will appreciate that for being equal to term " base pair ".

" polypeptide " is the polymer that amino acid residue is formed by connecting by peptide bond, can be natural or synthetic generation.The polypeptide that is less than approximately 50 amino acid residues is commonly referred to " oligopeptide ".

Term " promoter " is used to represent to provide in conjunction with RNA polymerase and start to be operatively connected the Gene Partial that comprises DNA sequence that coded sequence is transcribed with regard to its meaning well known in the art in this article.Promoter sequence is conventionally in 5 ' noncoding region of gene.

" protein " is the macromole that comprises one or more polypeptide chain.Protein also can comprise non-peptide composition, as glycosyl group.Sugar and other non-peptide substituent group can join in protein by producing protedogenous cell, and it can change with cell type.Herein at its amino acid backbone structure aspects definition albumen; Substituent group for example glycosyl is not specified conventionally, but can exist.

The sequence that term under fusion rotein background of the present invention " substantially similar " indication polypeptide comprises and canonical sequence (as the GP-Fc of this paper example merges) have at least 90% on over 10-20 amino acid whose comparison window, preferably at least 95% sequence homogeny.Sequence homogeny percentage ratio is measured by contrast two kinds of best aligned sequences in comparison window, and wherein in comparison window, the part of polynucleotide sequence and reference sequences (containing adding or disappearance) are compared to comprise and added or disappearance (being breach) is compared described two kinds of sequences with the best.This percentage ratio calculates as follows: the positional number that occurs identical nucleic acid base or amino acid residue by measuring in two kinds of sequences produces matched position number, sum by this matched position number divided by position in comparison window, the result obtaining is multiplied by 100 generation sequence homogeny percentage ratios.

Brief Description Of Drawings

Fig. 1 is schematic diagram and the purification of ZEBOVGP-Fc and FLAG-Fc albumen.A) schematic diagram of fusion rotein.Described ZEBOVGP-Fc fusion rotein comprises ZEBOV GP extracellular domain, and described extracellular domain has FLAG peptide tag at C-terminal, and is fused to human IgG1's hinge HeFc district.The FLAG label that described FLAG-Fc fusion rotein comprises the hinge HeFc district that is fused to human IgG1.B) SDS-PAGE of fusion rotein analyzes.The ZEBOVGP-Fc of protein A purification and FLAG-Fc goods are analyzed and use Coomassie blue stain in 4-12% gradient glue by degeneration SDS-PAGE.C) the Western engram analysis of ZEBOVGP-Fc and FLAG-Fc.Albumen decomposes by SDS-PAGE under Denaturing, transfers on pvdf membrane, and surveys with the anti-GP1mAb13F6-1-2 of ZEBOV specificity, anti-Flag M2mAb or the anti-human Fc Ab of goat.ZEBOV GP1, GP2-FLAG-Fc and FLAG-Fc band are indicated with arrow.Albumen and molecular weight marker thing are indicated with kDa.

Fig. 2 is the evaluation of ZEBOVGP-Fc fusion rotein.A) FPLC of the ZEBOVGP-Fc of protein A purification analyzes.(blueness) ZEBOVGP-Fc of indigested (Lycoperdon polymorphum Vitt) or enterokinase digestion passes through Superdex200 size-exclusion column under non-Denaturing, and the component of 38 collections is respectively recorded to 280nm absorbance.The peak arrow labelling that represents ZEBOVGP-Fc, ZEBOV GP and Fc.The peak of about 150kDa represents the carrier protein in enterokinase goods.Arrow shows the movement of molecular weight standard, and its molecular weight represents with kDa.B) the Western engram analysis of the ZEBOVGP-Fc albumen of enterokinase digestion.Gel filtration peak component (20 and 32) separately, is transferred to pvdf membrane in degeneration SDS-PAGE, with the anti-GP1mAb13F6-1-2 of ZEBOV specificity or the anti-human anti-Fc antibody of goat, surveys.(D) ZEBOVGP-Fc and the FLAG-Fc (Fc) that in gel, comprise indigested (U) or enterokinase digestion serve as a mark.The movement of arrow indication GP1, GP2-FLAG-Fc and FLAG-Fc.The movement of molecular weight marker and its size are indicated with kDa.

Fig. 3 has shown the analysis of inoculating anti-ZEBOV GP antibody in C57BL/6 mice.A) by the anti-ZEBOV GP specific antibody of virion ELISA, analyze.ZEBOVGP-Fc or FLAG-Fc inoculation for mice, and within after final inoculation 2 weeks, obtain blood serum sample.The titration serum on 96 orifice plates of radiation ZEBOV granule, rVSV-ZEBOVGP or contrast wild type VSV coating of purifying with sucrose.The terminal dilution of each mice serum is tired and is represented with point.B) facs analysis that mice serum is combined with the expressed ZEBOVGP in HEK293-ZEBOVGP cell surface.The goat anti-mouse IgG Ab dyeing of the HEK293 of HEK293-ZEBOVGP or control vector transfection mice serum (1 μ l) and PE coupling for cell, and pass through flow cytometry analysis.The block diagram of each mice serum shows with different colours, and the block diagram of the anti-GP mAb13F6-1-2 of positive control represents with Lycoperdon polymorphum Vitt dotted line.In order to make figure simple, the serum same color to two different mices (inoculating FLAG-Fc for another (right figure) inoculation for one with ZEBOVGP-Fc (left figure)).

Fig. 4 shows with the rVSV-ZEBOVGP neutralization of inoculating C57BL/6 mice serum.In Vero E6 cell, use the rVSV-ZEBOVGP of 100pfu to carry out plaque and reduce test, 5 times of dilute serums of ZEBOVGP-Fc or FLAG-Fc Mice Inoculated are processed for described rVSV-ZEBOVGP.Calculate the percent neutralization of each mice serum, as the plaque number of comparing untreated rVSV-ZEBOVGP, decline.Data show the average percent neutralization in the repeat samples of each mice serum dilution, and bar line represents standard deviation.

Fig. 5 shows with the humoral immune reaction in the BALB/c mouse of ZEBOVGP-Fc inoculation.4 ZEBOVGP-Fc or FLAG-Fc immunity for BALB/c mouse.Within finally after inoculation 2 weeks, collect blood serum sample.A) according to completing anti-ZEBOV GP specific antibody by virion ELISA described in Fig. 3 A on the flat board by rVSV-ZEBOVGP coating, analyze.The rVSV-ZEBOVGP neutralization of the BALB/c mouse serum of B) inoculating with ZEBOVGP-Fc or FLAG-Fc.The average neutralization of 1/10 dilute serum is by using the plaque minimizing test of repeat samples as described in Figure 4 to measure.The average percent (n=4) of average neutralization in each sample of data show, and bar line represents standard deviation.Difference between ZEBOVGP-Fc-and the neutralization of FLAG-Fc-Mice Inoculated serum is statistically significant (* *, p≤0.01).

Fig. 6 shows with the cell immune response in the BALB/c mouse of ZEBOVGP-Fc inoculation.In Fig. 5, through inoculation BALB/c mouse splenocyte, within 8 days after final immunity, collect, with fusion rotein or peptide, stimulate, and surperficial with the anti-CD8mAb staining cell of APC labelling, and carry out cell inner dyeing with the anti-IFN-γ mAb of PE labelling.Generate the CD8 of IFN-γ ⁺t cell percentage ratio passes through CD8 ⁺the cells were tested by flow cytometry of splenocyte gate.A) dot map analysis is used by oneself ZEBOVGP-Fc or FLAG-Fc inoculation and the CD8 of the representative mice that stimulates with ZEBOVGP-Fc or FLAG-Fc ⁺splenocyte.The two positive splenocyte of expressing CD8 and IFN-γ shows in rectangular box, and number represents the percentage ratio of IFN-γ in box-positive CD8+ cell.B-E) use by oneself ZEBOVGP-Fc (B, D) or FLAG-Fc (C, E) inoculation and with IFN-γ-positive CD8 in the mouse boosting cell of Fc fusion rotein (B, C) or synthetic peptide (D, E) stimulation of histogram graph representation ⁺the percentage ratio of cell.Fc fusion rotein ZEBOVGP-Fc or FLAG-Fc or ZEBOV GP specificity or the stimulation of contrast synthetic peptide for splenocyte.Data show is expressed the average percent (n=4 mice) of total CD8+ cell of IFN-γ, and bar line represents standard deviation.IFN-γ-positive CD8 in the ZEBOVGP-Fc-Mice Inoculated stimulating with ZEBOVGP-Fc and FLAG-Fc ⁺splenocyte percentage difference has significance,statistical (*, p=0.01).

Fig. 7 has shown the ZEBOVGP-Fc protection mice for the lethal attack of ZEBOV.ZEBOVGP-Fc for mice (solid triangle) or FLAG-Fc (open squares) inoculation, and finally inoculate latter 2 weeks, with the mice adaptability ZEBOV of 1,000pfu, attack.Survival percentage result mapping (every group of n=8 mice) to each inoculation group.

Fig. 8 has shown the experimental result with virion ELISA in the guinea pig serum of ZEBOVGP-Fc fusion rotein inoculation.QS-21 adjuvant inoculation containing ZEBOVGP-Fc for 7 Cavia porcelluss (GP-Fc1-GP-Fc7).Animal blood-letting after strengthening for 2 times, and by the anti-ZEBOVGP antibody of virion elisa assay serum.All animals develop the anti-ZEBOVGP antibody of high-titer.

Fig. 9 has shown the experimental result with virion ELISA in the guinea pig serum of contrast Fc fragment inoculation.The QS-21 adjuvant inoculation that only contains Fc fragment for 7 Cavia porcelluss (Fc1-Fc7).Animal blood-letting after strengthening for 2 times, and by the anti-ZEBOVGP antibody of virion elisa assay serum.Do not have animal to develop anti-ZEBOVGP antibody.

Figure 10 has shown the Zaire Ebola pseudotype virus neutralization by ZEBOVGP-Fc inoculation guinea pig serum.The false type of Zaire Ebola virus is used from inoculation ZEBOVGP-Fc or the serum neutralization of the Cavia porcellus of Fc fragment separately.Compare preimmune serum, measure percent neutralization.All ZEBOVGP-Fc inoculation animals develop anti-ZEBOV neutralizing antibody, and do not have Fc inoculation animal to develop anti-ZEBOV neutralizing antibody.Figure 11 shows the analysis of anti-Ebola virus antibody in three macaques.Before inoculation, the serum of three macaques is tested the GP antibody of anti-Ebola virus by virion ELISA.Monkey is not anti-Ebola virus antibody all.

Figure 12 has shown with the macaque of ZEBOVGP-Fc inoculation and has developed anti-Ebola virus antibody.The serum of 2 monkeys that inoculate with ZEBOVGP-Fc (GP-Fc1 and GP-Fc2) comprises the anti-Ebola virus antibody of the high-titer of being evaluated as virion ELISA, and only uses the serum of the contrast monkey of Fc fragment (Fc) inoculation not develop anti-Ebola virus antibody.

Figure 13 shows the Ebola's pseudotype virus neutralization with ZEBOVGP-Fc inoculation serum of macaque.With the macaque GP-Fc1 of ZEBOVGP-Fc inoculation and-2 and the serum of the contrast monkey Fc that only inoculates by Fc fragment in 96 orifice plates that comprise Vero E6 cell monolayer, by endpoint dilution assay, test anti-Ebola virus neutralizing antibody.Within after initial immunity (vaccine) 3 weeks, collect serum, animal is strengthened by corresponding virus, and strengthens within 3 weeks, collecting serum after (strengthening for the first time).Compare preimmune serum, measure percent neutralization.The monkey of ZEBOVGP-Fc inoculation develops anti-Ebola virus neutralizing antibody, and does not develop neutralizing antibody with the monkey of Fc contrast inoculation.

Detailed Description Of The Invention

The present invention has shown that the fusion rotein that comprises Filovirus glycoprotein (GP) fragment that is fused to immunoglobulin polypeptides fragment has experienced shearing and the processing of observing in natural GP.The result of demonstration is clear herein points out that described GP fusion rotein does not need viral vector or assembling assembly virus-like particle, itself is just enough to induce the protection that Filovirus is infected, and as the safety for Filovirus infection and effective subunit vaccine.

As below in detail as shown in, Filovirus GP (as the ZEBOV GP) ectodomain that is fused to immunoglobulin fragment (as human IgG1's Fc fragment) of expressing in mammalian cell experiences complicated post translational modification, comprises furin cutting and the homotrimer in natural GP, observed and forms.With the fatal dose that the inoculation of Filovirus GP-IgG fusant can protect mice to resist Zaire EBOV, attack.Result described herein shows that described Filovirus GP-IgG fusant does not need viral vector or assembling assembly virus-like particle; just be enough to induce the protection that Filovirus is infected, and shown that Filovirus GP-IgG fusant is as the safety infecting for Filovirus and effective subunit vaccine.

Filovirus GP-domain-immunoglobulin fusion proteins

Ebola virus and Marburg (Marburg) virus forms Filoviridae (Filoviridae).There are 5 kinds of Ebola virus: Zaire's (type sepecies), the Sudan, Christopher Eccleston, Ben Dibujiao and the Ivory Coast.There is a kind of Marburg virus.Shown in glycoprotein (GP) be the unique structural protein that form virion protrusion of surface, described projection regulates virus to enter susceptible host cell by receptors bind.GP is the Filovirus albumen of most study, not only due to its importance in cell entry and pathogenesis, and because is the primary target of vaccine development.

Filovirus granule comprises the long strand RNA genome of about 19kb, and 7 structural protein and 1 non-structural protein (Volchkov etc., Adv Virus Res2005 encode; 64:359-81).Envelope glycoprotein (GP) is present in virion protrusion of surface, and to receptors bind, cell entry and immunity work (Feldmann etc., J Gen Virol2001 December; 82 (Pt12): 2839-48 and Takada etc., Proc Natl Acad Sci U S A1997 December 23 days; 94 (26): 14764-9).Described Filovirus GP integrates glycoprotein from 1 type film of gene 4, and experience relates to the N-end of GP and the complicated processing that closely between membrane portions, furin cuts and disulfide bond forms.The ripe cross-film GP existing on the film of peplos and infection cell is formed by two subunits: by the covalently bound film grappling GP2 to GP1N end of disulfide bond; it comprises the glycosylated mucin spline structure of high o-territory (Volchkov etc., Proc Natl Acad Sci U S A1998 May 12; 95 (10): 5762-7) and Jeffers etc., J Virol2002 December; 76 (24): 12463-72).A large amount of GP1 is coming off from GP2 subunit discharges in cell.In addition; lack cross-film anchor with the total amino acid whose non-structure soluble sugar albumen of amino terminal 295 (sGP) of GP1; and form the homodimer that disulfide bond connects, Hemapoiesis (Feldmann etc., Curr Top Microbiol Immunol1999 that it is infected by EBOV rather than MARV; 235:1-21).

A lot of genes of coding Filovirus GP are cloned, and are described in document (seeing for example WO2006/037038).Technical staff can easily clone required gene or use aforementioned gene.Table 1 provides the GenBank accession number from the exemplary GP sequence of various Ebolas and Marburg virus hypotype to gather.

Table 1

In fusion rotein of the present invention, described GP albumen can be full-length proteins or its fragment, for example GP ectodomain.Described ectodomain can be available from various sources.For example, it can be from the Filovirus glycoprotein of natural generation, the Filovirus glycoprotein of sudden change (as comprising simple point mutation, the deletion of one or more aminoacid, one or more domain as the albumen of mucin domain deletion), or the chimeric Filovirus GP albumen of the domain that comprises different Filovirus strains, species or genus.As mentioned above, well identified the GP albumen of Ebola virus, and technical staff can easily identify the suitable construction territory in the GP albumen of particular separation thing.For example, in following exemplary GP, use the fragment (the residue 1-637 of peptide sequence shown in GenBank accession number U23187) that comprises ectodomain.The sequence alignment technology that use is known, technical staff can easily identify the respective segments from the GP of other separators.

The immunoglobulin fragment of fusion rotein of the present invention can be from the antibody of any kind: IgA, IgD, IgE, IgG or IgM.In common embodiment, use human IgG polypeptide.As mentioned above, IgG molecule is divided into 4 subclass that are called IgG1, IgG2, IgG3 and IgG4.The DNA sequence of encoding human and non-human immunoglobulin chain is well known.

In conventional embodiment, use immunoglobulin heavy chain constant region.As mentioned above, in the example of IgG molecule, CH comprises C _h1, C _h2 and C _h3 domains and hinge region.In some embodiments, use Fc fragment.As mentioned above, in IgG, IgA and IgD, the corresponding C in described Fc district _h2 and C _h3 domains and hinge region.At IgM and IgE, described Fc district comprises three CH (C _h2, C _h3 and C _h4).Described hinge, as the flexible introns between two parts of fusion rotein, makes the each several part independent action of molecule.

Know the fusion rotein preparation that comprises immunoglobulin heavy chain constant region.A lot of examples of this class fusant are described in document (referring to for example U.S. Patent number 7,754,855; 5,480,981; 5,723,125,5,808,029; WO97/23614; WO98/28427, and reference by reference).Fc fusion rotein can comprise variation Fc molecule (as being described in U.S. Patent number 7,732,570).

Described Filovirus GP fragment and immunoglobulin fragment can be connected to each other with variety of way well known by persons skilled in the art.For example, described fusion rotein Filovirus GP segment composition is to immunoglobulin fragment, and the C-end of wherein said Filovirus GP fragment is connected on the N-end of immunoglobulin fragment.

In some embodiments, described fusion rotein can also comprise two peptide linkers between fragment.For example, the C-end of described Filovirus GP can be connected to by peptide linker the N-end of described immunoglobulin fragment.Conventionally, described peptide linker is immunology inertia, and is designed to suitably to process Filovirus GP.Described peptide linker for example can comprise the cutting site of protease for example or sequence, to help the separation of fusion rotein, epitope tag.Design and use joint peptide is well known.

The present invention has also comprised the nucleic acid molecules of code book invention fusion rotein.Nucleic acid molecules of the present invention can be rna form or the DNA form generating that clone obtains or synthetic.Described DNA can be two strands or strand." separation " nucleic acid molecules refers to the nucleic acid molecules shifting out from its natural surroundings, DNA or RNA.For example, the contained recombinant DNA molecules of carrier is regarded as with regard to the object of the invention separated.The DNA molecular solution that comprises the recombinant DNA molecules that is held in heterologous host cell or purification (part or substantially) through other example of DNA isolation molecule.Separated RNA molecule comprises in the body of DNA molecular of the present invention or external rna transcription thing.According to the present invention, separated nucleic acid molecules also comprises synthetic this quasi-molecule producing.

Nucleic acid molecules of the present invention comprises the DNA molecular of the open reading frame (ORF) that contains code book invention fusion rotein, with the DNA molecular comprising with this paper institute those essentially identical sequences of example, but described sequence is due to the gene code degeneracy fusion rotein of the present invention of still encoding.Therefore,, in common embodiment, described nucleic acid molecules is for expressing the expression vector of fusion rotein of the present invention.The nucleic acid molecules of coded polypeptide can be operatively connected controls the adjusting sequence that expression vector transcription is expressed, and then introduces in host cell.Except transcriptional regulatory sequences for example promoter and enhancer, expression vector can comprise transcriptional regulatory sequences and be applicable to selecting being loaded with the marker gene of the cell of expression vector.

Fusion rotein polypeptide of the present invention can generate according to conventional art in recombinant host cell.Fusion rotein of the present invention can be expressed in various host cells well known to those skilled in the art.For example, described albumen can be expressed in mammalian cell.Suitable mammalian host cell comprises African green monkey kidney cell (as Vero; ATCC CRL1587), human embryonic kidney cell is (as 293-HEK; ATCC CRL1573), baby hamster kidney cell is (as BHK-21, BHK-570; ATCC CRL8544, ATCC CRL10314), Madin-Darby canine kidney(cell line) is (as MDCK; ATCC CCL34), Chinese hamster ovary cell is (as CHO-K1; ATCC CCL61; CHO DG44), rat pituitary cell is (as GH1; ATCC CCL82), HeLa S3 cell (as ATCC CCL2.2), rat hepatoma cell are (as H-4-II-E; ATCC CRL1548), SV40-transforms monkey-kidney cells (as COS-1; ATCC CRL1650) and Mus embryonic cell (as NIH-3T3; ATCC CRL1658).

For mammalian hosts, transcribe and translate conditioning signal and can originate from mammalian virus, such as adenovirus, bovine papilloma virus, simian virus etc., wherein said conditioning signal is relevant with the specific gene of high level expression.Suitable transcribe and translate regulate the sequence also can be available from mammalian genes, for example actin, collagen, myosin and metallothionein gene.

Expression vector can be used various standard techniques to introduce in mammalian host cells, and described technology comprises the sending of calcium phosphate transfection, liposome-mediated transfection, microparticle bombardment mediation, electroporation etc.Described transfectional cell can be selected and breed to provide to comprise stable integration to the recombinant host cell of the expression vector of host cell gene group.Carrier is introduced to eukaryotic technology and used dominant selectable marker to select the technology of this stable conversion body is well known.It is well known using transgenic animal expressing heterologous albumen.Recombinant DNA is introduced to animal (as mammal) germ cell can use the many standard techniques in transgenic animal technology to complete.Referring to such as Hogan etc.; Manipulating the Mouse Embryo:A Laboratory Manual (< < mice embryonic operation: laboratory manual > >); the publishing house of cold spring harbor laboratory at cold spring port, New York (Cold Spring Harbor Laboratory Press), 1986; With U.S. Patent number 5,811,634.Once recombinant DNA is incorporated in ovum, the described ovum short time cultivates, and is then transferred in the pseudo-fetus animal of the same species that obtains ovum.And whole animal capable is as the expression system of fusion rotein of the present invention, common described fusion rotein accumulates in animal product, therefrom can not damage animal and gathers in the crops albumen.In a preferred embodiment, described fusion rotein accumulation in milk, ovum, hair, blood or urine.If described fusion rotein accumulates in the milk of animal, suitable mammal is ruminant, ungulate, domestic mammal and dairy stock.Particularly preferred animal is goat, sheep, camel, cattle, pig, horse, bull and yamma.Know the method for gathering the transgenic cow of recombinant peptide in its milk that is created on: referring to Newton (1999, J.Immunol.Methods231:159-167), Ebert etc. (1991, Biotechnology9:835-838) and U.S. Patent number 6,210,736,5,849,992,5,843,705,5,827,690,6,222,094.

Fusion rotein of the present invention also can be expressed in other higher eucaryotic cells, for example birds, insecticide, fungus, yeast or plant cell.Described rhabdovirus system provides the effective ways of clone gene being introduced to insect cell.Comprising yeast cells also can be for expressing gene described herein interior fungal cell.Interested especially yeast specie comprises saccharomyces cerevisiae (Saccharomyces cerevisiae), pichia pastoris phaff (Pichia pastoris) and pichia methanolica (Pichia methanolica) in this respect.The promoter that the suitable promoter of expressing in yeast comprises GAL1 (galactose), PGK (phosphoglyceric kinase), ADH (alcoholdehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinol dehydrogenase) etc.Expression vector also can be introduced whole plant (as tobacco plant), plant protoplast, complete plant tissue or separated plant cell.The method of expression vector introduced plant tissue is comprised to direct infection or plant tissue and Agrobacterium tumefaciems (Agrobacterium tumefaciens) is cultivated altogether, the sending of microparticle bombardment mediation, DNA injection, electroporation etc.

Or the gene of code book invention polypeptide can be expressed in prokaryotic host cell.Suitable prokaryotic hosts comprises escherichia coli (E.coli) and bacillus subtilis (Bacillus subtilis).Suitable e. coli strains comprises BL21 (DE3), BL21 (DE3) pLysS, BL21 (DE3) pLysE, DH1, DH4I, DH5, DH5I, DH5IF', DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451 and ER1647 are (referring to for example Brown (volume), Molecular Biology Labfax(< < molecular biology experiment handbook > >) (academic press (Academic Press) 1991)).Suitable bacillus subtilis bacterial strain comprises BR151, YB886, MI119, MI120 and B170 (referring to for example Hardy, " Bacillus Cloning Methods(bacillus cereus cloning process) ", include the DNA clone in DNA Cloning:A Practical Approach(< <: hands-on approach > >), Glover (volume) (IRL publishing house (IRL Press) 1985)).

In prokaryotic hosts, the method for expressing protein is known (referring to such as Williams etc. for those skilled in the art, " Expression of foreign proteins in E.coli using plasmid vectors and purification of specific polyclonal antibodies(is used plasmid vector to express the purification of foreign protein and specific polyclonal antibody in escherichia coli) ", include in DNA Cloning2:Expression Systems(< < DNA clone 2: expression system > >), the 2nd edition, Glover etc. (volume), the 15th page (Oxford University Press (Oxford University Press), 1995), Ward etc., " Genetic Manipulation and Expression of Antibodies(genetic manipulation and antibody expression) ", include the monoclonal antibody in Monoclonal Antibodies:Principles and Applications(< <: principle and application > >), the 137th page of ((Wiley-Liss of this company of Willie, Inc.), 1995), and Georgiou, " expression of albumen in Expression of Proteins in Bacteria(antibacterial) ", include the protein engineering in Protein Engineering:Principles and Practice(< <: principle and put into practice > >), Cleland etc. (volume), the 101st page of ((the John Wiley & Sons of John Wei Lisen company, Inc.), 1996)).

Once express, fusion rotein of the present invention can, according to this area standard method purification, comprise ammonium sulfate precipitation, fractionation column chromatograph, gel electrophoresis etc.

Immunogenic composition

Purification of the present invention or partially purified fusion rotein can be made vaccine (also referred to as " immunogenic composition ") according to method well known in the art.Some compositions can comprise adjuvant to strengthen immunoreation.In described preparation, the optimal ratio of each component can pass through technical measurement well known to those skilled in the art.Described immunogenic composition can give patient according to technology well known to those skilled in the art.Described patient can be people, non-human animal (as cattle, horse, pig, Canis familiaris L. and cat).

The preparation of this based composition and use are well known to those skilled in the art.Composition of liquid medicine comprises liquid-carrier conventionally, for example water, oil, animal or plant oil, mineral oil or synthetic oil.Can comprise physiological solt solution, glucose or other sugar juice or glycerol, for example ethylene glycol, propylene glycol or Polyethylene Glycol.

The polynucleotide of code book invention fusion rotein also can give patient.Conventionally, preparation is adapted at driving in people's cell the expression cassette of expression.This method is described in for example Wolff (1990) Science247:1465-1468; U.S. Patent number 5,580,859 and 5,589,466.

Described compositions is applicable to single give or series gives.When series gives, give the follow-up inoculation of initial administration to strengthen immunoreation, and be commonly referred to enhancing inoculation.The present composition can just be exempted from the enhancing composition that compositions is just exempted from by antigen as any various differences of use, or as just exempting from compositions.Therefore, one aspect of the present invention provides induction and/or has strengthened the method to antigen immune reaction in individuality.

The time that gives enhancing composition is known for those skilled in the art.Conventionally in the several weeks or the several months that give just to exempt from after compositions, give enhancing composition, for example about 2-3 week or 4 weeks or 8 weeks or 16 weeks or 20 weeks or 24 weeks or 28 weeks or 32 weeks.

The present composition can comprise other Filovirus antigen, or just exempts from or strengthen inoculation to comprise other antigens.Inessential to the present invention with other antigens of fusion rotein coupling of the present invention, and can be for example Filovirus antigen, the nucleic acid of expressing it, virus-like particle (VLP) or the replication defect type that comprises nucleic acid or replicating vector, for example prior art vaccine discussed above.This viral vector for example comprises adenovirus vector, vaccinia virus vector, fowl pox carrier as bird pox and canary pox, herpesvirus vector, vesicular stomatitis virus carrier or α viral vector.Technical staff should be appreciated that immunogenic composition of the present invention can comprise plurality of antigens and carrier.

Respectively just exempt from antigen (yet using a lot of enhancing composition) in enhancing composition do not need consistent, but should share epi-position.Described antigen can corresponding target pathogen or cell in complete antigen, or its fragment.Can use the artificial string (string) of peptide epitopes or epi-position, more effectively excise the coded sequence in unwanted protein sequence and a kind of or a plurality of carrier in antigen.For example, can comprise one or more other epi-positions, for example the epi-position, the particularly epi-position of different HLA type individual identification of t helper cell identification.

As mentioned above, immunogenic composition of the present invention can comprise adjuvant.The adjuvant that is applicable to jointly giving according to the present invention should be potential safety, well-tolerated and effective in people, comprise QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alumen and MF59 (referring to Kim etc., 2000Vaccine18:597, and list of references wherein).

Other adjuvants that can give comprise agglutinin, somatomedin, cytokine and lymphokine, for example alpha-interferon, IFN-γ, platelet derived growth factor (PDGF), granulocyte colony-stimulating factor (gCSF), granulocyte macrophage colony stimulating factor (gMCSF), tumor necrosis factor (TNF), epidermal growth factor (EGF), IL-I, IL-2, IL-4, IL-6, IL-8, IL-IO and IL-12, or its code nucleic acid.

As mentioned above, the present composition can comprise pharmaceutically acceptable excipient, carrier, buffer, stabilizing agent or other materials well known to those skilled in the art.These materials should be nontoxic, do not answer the effect of interferon activity composition.The precise nature of carrier or other materials depends on route of administration, for example (as intestinal), intranasal, intramuscular or intraperitoneal approach in oral, intravenous, percutaneous or subcutaneous, mucosa.Administration is Intradermal normally, for example subcutaneous or intramuscular.

The intramuscular administration of immunogenic composition can realize with the suspension that pin is injected fusion rotein or its code nucleic acid.Or use not must (needless) injection device to give above-mentioned composition (using for example Biojector (TM)) or the lyophilized powder that comprises vaccine.

For intravenous, percutaneous or subcutaneous injection, or in patient part injection, fusion rotein can be the acceptable aqueous solution form of parenteral, and it is not containing pyrogen and have suitable pH, isotonicity and stability.Those skilled in the art can utilize, and such as waiting, ooze supporting agent and prepare suitable solution as sodium chloride injection, ringer's injection, lactated Ringer's injection.As needs, can comprise antiseptic, stabilizing agent, buffer, antioxidant and/or other additives.Also can use slow releasing preparation.

Conventionally, administration has prevention object, to infect or the immunoreation of paired Filovirus antigen in previous existence appears in symptom.The disease that can treat or prevent according to the present invention or disorderly comprise wherein immunoreation and can play those of protection or therapeutical effect.In other embodiments, can give described Filovirus glycoprotein Fc fusion rotein for post-exposure prophylaxis.

Give the immunogenic composition that object comprises fusion rotein or its coded polynucleotide, in object, generate anti-Filovirus immunoreation.Being enough to induction can detect immunoreactive amount of composition and be defined as " immune effective dose ".As follows, immunogenic composition induction antibody response of the present invention, and CD8+T cell immune response.In common embodiment, immunoreation is protective immunological reaction.

The actual amount giving and medicine-feeding rate and time-histories depend on characteristic and the seriousness that will treat.Prescribed treatment, as determined dosage etc., is those of ordinary skill and other doctors or the Chinese veterinarian's of veterinary responsibility, and the disease of the disease that consideration will be treated conventionally, individual patient, sends site, medication and other factors known to the skilled.The example of above-mentioned technology and scheme can be referring to Remington's Pharmaceutical Sciences (< < Lei Mingdun pharmaceutical science > >), the 16th edition, Osol, A. compiles, and 1980.

After the optional preparation of Filovirus glycoprotein Fc fusion rotein and this granule generates in compositions, described fusion rotein can give individuality, particularly people or other Primates.Can give people, or another kind of mammal, for example mice, rat, hamster, Cavia porcellus, rabbit, sheep, goat, pig, horse, cattle, donkey, Canis familiaris L. or cat.Being delivered to non-human mammal need to be for therapeutic purposes, but can be for Experimental Background, for example research to the immune response mechanism of fusion rotein.

In an exemplary arrangement, with the dosage of 10 microgram-1 microgram/injections, give (as intramuscular) fusion rotein.Can after 4 weeks, strengthen at as many as (boost).If need, said composition can be packed in medicine box, packing or the allotter that can comprise one or more unit dosage forms that contain active component.For example, described medicine box can comprise metal or plastic tab, as blister package.Described medicine box, packing or allotter can have administration description.

The present composition can be individually dosed or with other treatment simultaneously or successively administering drug combinations, this depends on the disease that will treat.

Detect immunoreation

Fusion rotein of the present invention infects for detection of Filovirus.In detection of biological sample, the method for body fluid or cell immune response is well known." biological sample " used herein is available from any lymphocyte of patient or the sample that comprises antibody.For example, described sample can be whole blood, expectorant, serum, blood plasma, saliva, cerebrospinal fluid or urine.Albumen of the present invention for following test for example to measure and the existing or lacking of the antigen reactive antibody of Filovirus or immunocyte (as lymphocyte, CD4+T-cell, CD8+T-cell, B-cell, NK cell, mastocyte, antigen-presenting cell).Exist described antibody or immunocyte to indicate the sensitization to Filovirus antigen that can indicate Filovirus to infect before.

Those of ordinary skill known various test models in capable territory are to be used one or more polypeptide to detect the antibody in samples.For example, described test can relate to the fusion rotein that is fixed on solid support with combination and remove the antibody in sample.Described test also can relate to by being combined in antibody on solid support or in conjunction with albumen prey fusion protein (as protein A, Protein G, protein A/G, Avidin, Streptavidin etc.).Described test can relate to micromolecule as biotin, enzyme or fluorogen labelling fusion rotein.Or, patient's antibody can be used in conjunction with albumen or type specific antibodies to solid phase (as anti-human IgM or IgG antibody), then with labelling or unlabelled fusion rotein (FiloGP-Fc), process, and finally use coupling antibody and/or in conjunction with albumen (as Avidin-FITC, Avidin-peroxidase etc.), detect the fusion rotein of combination.Described fusion rotein can be separated or at recombinant virus (as herpes Stomatovirus) surface expression.Then described binding antibody can be used the detection agent that comprises reporter group to detect.The antibodies that suitable detection agent comprises is to solid phase fusion rotein or antibody/polypeptide complex, or with the free polypeptide (as in half competitive trials) of reporter group labelling.Or, can use competitive trials, be wherein attached to the antibody reporter group labelling of polypeptide, and after antigen and sample hatch, it be attached on immobilized antigen.The degree indication sample that sample composition inhibition traget antibody is combined with polypeptide and the reactivity of immobilized polypeptide.In some embodiments, described test is enzyme-linked immunosorbent assay (ELISA), chemoluminescence test or fluorescent test.

Detecting the method for cell immune response also knows.In this test, antigen-presenting cell, immunocyte are (as CD8 ⁺t-cell, CD4+T-cell, B-cell etc.) and fusion rotein of the present invention under appropraite condition, hatch, to induce the propagation of Filovirus protein-specific immune effector cell or activation (as CD8 ⁺t – cell, CD4+T-cell, B-cell, NK and iNKT cell etc.).Induction of immunity effector lymphocyte's (as helper T-cell, CTL, B-cell, NK, NKT etc.) antigenic specificity reaction can be by a lot of technology (as passed through ⁵¹cr release test, ³h-thymidine mixes, CFSE and other facs analysis proliferation tests, intracellular cytokine dyeing and facs analysis, ELISPOT, secrete cytokines be as the detection of INF-γ) detect.

In some embodiments, the ability infecting with Filovirus in antibody in energy specimen.This can use, and for example the plaque based on expressing the recombinant virus neutralization of Filovirus GP reduces test or endpoint dilution assay carries out.Equally, neutralization can be measured by ELISA, microscopic analysis or facs analysis dyeing infection cell, with the antiviral antibody of coupling micromolecule, enzyme or fluorogen, and/or second antibody or with the combination albumen (as Avidin) of enzyme or fluorogen coupling.Or the neutralization of the recombinant virus of expressing Filovirus GP and comprising fluorescence protein gene (as GFP, YFP etc.) can be measured by the minimizing in fluorescence infection cell with fluorescence microscope analysis or facs analysis.For example, the recombinant vesicular stomatitis virus (VSV) of expression Filovirus GP can be used for this test.As follows, because the neutralizing antibody for Filovirus points to the only envelope glycoprotein in GP(virocyte surface), the neutralization of Filovirus is simulated in the neutralization of expressing the restructuring VSV of this albumen.The coated plate of VSV granule ELISA of also expressing the recombinant vesicular stomatitis virus (VSV) of Filovirus GP by use detects whole antibody.The method of carrying out neutralization test is known for those skilled in the art.Exemplary test is described below.

Described the present invention, in explanation mode, provide the following example, and be not construed as limiting.

Embodiment

Provide following examples to be illustrative rather than definitive thereof the present invention of prescription.

Embodiment 1

1. materials and methods

1.1. cell line

Lack Chinese hamster ovary (CHO) cell of enzyme dihydrofolate reductase (dhfr-) available from American type culture collection (American Type Culture Collection); and amplification (Silberstein etc., J Virol2001 January in the Yi Kefushi that comprises 10% hyclone (FBS) (Iscove) culture medium; 75 (2): 717-25).Human embryo kidney HEK293-H cell (hero company (Invitrogen)) maintains in being supplemented with the Da Erbaikeshi improvement Yi Geershi culture medium (DMEM) of 10%FBS.Vero E6 cell is grown in being supplemented with the Yi Geershi minimum essential medium (MEM) of 10%FBS.Baby hamster kidney cell (BHK-21) maintains in the DMEM culture medium that is supplemented with 5%FBS.BSR-T7 cell is the BHK-21 cell of expressing phage t7 RNA polymerase (Buchholz etc., J Virol1999 January; 73 (1): 251-9), described cell is provided by doctor's K.Conzelmann (Pei Tengke Fil institute (Pettenkoffer Institute) of Munich, Germany) friendship, and maintains (hero company) in the DMEM culture medium that is supplemented with 5%FBS and 1mg/ml Geneticin.

1.2. mice

C57BL/6 and BALB/c mouse be available from National Cancer Institute (National Cancer Institute), Frederick Taylor cancer research and centre of development (Frederick Cancer Research and Development Center) (Maryland State Frederick Taylor).All mices are raised in micro-isolator cage, and standard rodent and the water of searching for food is arbitrarily provided.Blood sample is available from lateral tail vein.Research is carried out with relating to zooperal federal regulations and stipulating about animal according to animal welfare method and other, and meets the laboratory animal nursing of the National Research Council in 1996 and the principle described in guide for use.Carrying out the facility of these researchs is all authorized by International Laboratory Animal assessment and certification committee.Described animal operation scheme is by CBER-FDA or USAMRIID animal care and use committee (IACUC) approval.

1.3. cloning process

Zaire Ebola virus in pVR-1012-ZEBOV-GP (ZEBOV) glycoprotein (GP); the cDNA of Mayinga strain (GenBank accession number AF272001) provides (Yang etc., Nat Med2000 August by the Gary doctor Nabel friendship at the NIH vaccine research center of Maryland State Bei Saisida; 6 (8): 886-9).Described glycoprotein gene has 8 adenosines (A) in the rna editing site that need to generate total length ZEBOV GP.Use standard gene engineering to build following plasmid:

1.3.1.pEF1-EBOV-GP

Described GP region is used NcoI and Asp718 (Roche Applied Science Fiction Co. (Rochee Applied Science)) Restriction Enzyme to shear from pVR-1012-ZEBOV-GP, with archaeal dna polymerase Klenow (New England's biology laboratory (New England Biolabs)) enzyme, fill to generate blunt ends, and be cloned into subsequently the EcoRV site of mammal expression plasmid pEF1/Myc-His-B (hero company).Gained plasmid is called pEF1-EBOV-GP.

1.3.2.pEF-ZEBOVGP-Fc

Plasmid for construction expression ZEBOV GP Fc fusion rotein, the PCR fragment (GenBank accession number U23187) of aminoacid 1 – 637 of coding ZEBOV GP extracellular domain increases from pVR-1012-ZEBOV-GP, synthetic oligonucleotide GP/SalI (5 '-GTCGACAGTATGGGCGTTACAGGAATATTGCAGTTA-3 ') is used in described amplification, SalI site before its coded sequence that comprises signal peptide GP, and GP/Flag/SpeI (5 '-ACTAGTACTCACCTCCCTTGTCATCGTCGTCCTTGTAGTCTCCACCGCCGTC CGGAAGGGTTTTATCAACAAA-3 '), it comprises SpeI site, then be artificial donor splicing site, the coded sequence of FLAG labelled peptide DTKDDDDK and the nucleotide of GP 1887 – 1911.This PCR fragment be cloned into replace ICAM1cDNA fragment and with SalI and the SpeI site of human IgG1 Fc fragment with pEF-ICAM5 (1-2) Fc of frame, Silberstein etc., J Virol2001, the same.Silent mutation (GTCGAC is to GTAGAC, and CTAGTT is to CTCGTT) is incorporated into GP coded sequence to eliminate inner SalI and SpeI restriction site.Gained plasmid is called pEF-ZEBOVGP-Fc.

1.3.3.pEF-FLAG-Fc

For the plasmid of construction expression human IgG1 Fc fragment, the ICAM1 sequence in pEF-ICAM5 (1-2) Fc replaces by the cDNA fragment of coding ZEBOV GP signal peptide and FLAG label.For this reason, the PCR fragment of amino acid/11-32 of amplification coding ZEBOV GP signal peptide, is used pVR-1012-ZEBOV-GP as nucleotide 73 – 96 and the SpeI restriction site of SP/Flag/SpeI (5 '-ACTAGTACTCACCTCCCTTGTCATCGTCGTCCTTGTAGTCTCCACCGCC-GG AAAATGTTCTTTGGAAAAGGAT-3 '), the GP cDNA of template and synthetic oligonucleotide GP/SalI (on seeing) and the FLAG label of encoding.SalI and the digestion of SpeI Restriction Enzyme for the PCR fragment of amplification, and be cloned into pEF-ICAM5 (1-2) Fc with same enzyme digestion.Gained plasmid is called pEF-FLAG-Fc.

1.3.4.pVSV-ZEBOVGP

The recombinant vesicular stomatitis virus (VSV) of deleting for the replication form VSV-G of construction expression ZEBOV GP (rVSV-ZEBOVGP), the PCR fragment (amino acid/11-676) of coding ZEBOV GP is used oligonucleotide GP/NheI (+) (5 ' ACTAGTAGTATGGGCGTTACAGGAATATTGCAGTTA-3 ') from pVR-1012-ZEBOV-GP, described oligonucleotide identical with GP/SalI (except replacing the SalI site of NheI restriction site), with antisense primer GP/NheI (-) (5 '-GCTAGCCTAAAAGACAAATTTGCATATACAGAA-3 ') amplification, described PCR fragment is sheared with NheI, and be cloned in the pVSV Δ G of NheI shearing.Gained plasmid is called pVSV-ZEBOVGP.

1.4 are chosen in HEK-293-H stable transfection of cell surface expression ZEBOV GP

According to manufacturer's suggestion, use pEF1-ZEBOV-GP or carrier pEF1 transfection HEK293-H cell for Fugene6 reagent (Roche Applied Science Fiction Co.), and select stable transfectant with 350 μ g/mL Geneticins, and be called HEK293-ZEBOVGP or HEK293 cell.Generate single cell clone, and use anti-ZEBOV GP monoclonal antibody (mAb) 13F6-1-2 to analyze (Wilson etc., Science2000 March 3 by flow cytometer; 287 (5458): 1664-6, Lee etc., J Mol Biol2008 January 4; 375 (1): 202-16) to be chosen in stable transfection of cell surface expression high level ZEBOV GP.

The generation of 1.5Fc fusion rotein and purification

Fc fusion rotein generates in Chinese hamster ovary celI transfectant.For this reason, 0.45 μ g pDHIP and 3.5 μ g pEF-ZEBOVGP-Fc or pEF-Fc use Fugene6 reagent cotransfection for CHO dhfr-cell, as being described in Silberstein etc. above, J Virol2001, the same.Simply say, cell transfecting is 37 ° of C growth 48h in the Yi Kefushi that is supplemented with hypoxanthine and thymidine, comprise 10%FBS (Iscove) culture medium, 1:10 division, and select in there is no Yi Kefushi (Iscove) culture medium containing dialysis FBS of fill-in.After within 14 days, selecting, by terminal dilution in 96 orifice plates, obtain single cell clone.Analyze the expressing fusion protein of the supernatant of 56 single cell clones, described analysis is by catching ELISA and dyeing with anti-GP mAb and HRP coupling goat anti-mouse antibody (Ab) in 96 orifice plates of the anti-human Fc antibody coating of goat.By progressively increasing the concentration of methotrexate (MTX), realize the overexpression of Fc fusion rotein.Cell reaches respectively the top level of ZEBOVGP-Fc and FLAG-Fc expression at 0.08 and 0.32 μ M MTX.

Fc fusion rotein is purification as previously mentioned: Silberstein etc., J Virol2001 is the same.Simply say, express the Chinese hamster ovary celI of recombiant protein and grow 3 days in comprising the Yi Kefushi of 10%FBS (Iscove) culture medium, with Yi Kefushi culture medium washing monolayer twice, and growth in serum-free OptiMEM culture medium (hero company).Supernatant is gathered in the crops 2-3 time with 24h interval, in 3,000xg clarification, and is stored in-20 ℃.Fc fusion rotein in OptiMEM supernatant is by the affinity chromatography purification of protein A-agarose column.Elution fraction is analyzed by degeneration sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the component that comprises Fc fusion rotein described in collecting, use Amicon ultra post (Millipore Corp. (Millipore)) concentrated, and wash with PBS.

1.6Western engram analysis

The albumen of purification separates in degeneration SDS-PAGE, transfer to Immobilon-P polyvinylidene fluoride (PVDF) film (Millipore Corp.), and with 1:1, the anti-FLAG M2mAb of the anti-ZEBOV GP13F6-1-2mAb of mice (USAMRIID), mice (sigma chemistry product company (Sigma Chemical Co.)) or mountain goat anti-human igg Fc Ab and the 1:3 of 000 dilution, 000 dilution through phosphatase enzyme mark goat anti-mouse or the anti-goat Ab of rabbit dyeing.5-bromo-4-chloro-3-indolylphosphate-nitrate blue tetrazolium substrate (KPL company (Kpl Inc.)) that Western trace is recommended with manufacturer.

1.7.rVSV-ZEBOVGP rescue

For generating the restructuring VSV that is loaded with ZEBOV GP (rVSV-ZEBOVGP), use is by the reverse genetics system of John doctor Rose (Yale University of New Haven, the Connecticut State) exploitation, and John doctor Rose friendship provides the plasmid [27] of VSV genome (pVSV Δ G), VSV nucleoprotein (pBS-N), VSV phosphoprotein (pBS-P) and VSV-L polymerase (pBS-L) that coding VSV full-length gene group [pVSVFL (+)], VSV-G delete.For this reason; BSR-T7 cell grows into 80-90% degrees of fusion in 6 orifice plates; with 0.25 μ g pBS-N, 0.6 μ g pBS-P, 0.13 μ g pBS-L and 1 μ g pVSV-ZEBOVGP or positive control pVSVFL (+) cotransfection; every hole is used 5ul liposome (Lipofectamine) 2000 (hero companies) as adjuvant (Lawson etc., Proc Natl Acad Sci U S A1995 May 9; 92 (10): 4477-81; Garbutt etc., J Virol2004 May; 78 (10): 5458-65).Hatch after 48h for 37 ℃, collect supernatant, and for infecting the BHK-21 cell of 50% fusion.After single-layer showing cytopathic effect (CPE), collect the supernatant that comprises rVSV-ZEBOVGP or VSV, titration in Vero E6 cell, and be stored in-80 ℃.

1.8.ZEBOVGP-Fc the enterokinase of albumen cutting

By processing the Fc fragment of removing ZEBOVGP-Fc with every mg fusion rotein 0.4 restricted protease enterokinase of μ g (New England Biolabs, Inc. (US) Massachusetts, United States of America), described enterokinase cuts FLAG peptide engineered between GP and Fc fragment.Described digestion product is analyzed by FPLC in Superdex200 post (GE health care companies (GE Healthcare)) is upper under non-Denaturing.Collected component is analyzed by degeneration SDS-PAGE with anti-ZEBOV GP mAb13F6-1-2 or anti-Fc Segment A b and the stain-fast Western trace of suitable phosphoric acids enzyme-coupling two.

1.9. inoculation and ZEBOV attack

The 100 μ g ZEBOVGP-Fc (n=8) or the inoculation of 100 μ g Fc (n=8) intraperitoneal (i.p.) that are dissolved in complete Freund's adjuvant for 6-8 C57BL/6 mice in age in week.Latter the 21st, 45 and 60 days of inoculation, animal is strengthened with the 25 μ g albumen that are dissolved in incomplete Freund's adjuvant.Before attacking, from each mice, obtain blood serum sample.Latter 2 weeks of final inoculation, with 1 of PBS dilution, 000pfu mice adaptability ZEBOV i.p. injects to attack mice (Bray etc., J Infect Dis1998 JIUYUE; 178 (3): 651-6; Bradfute etc., J Immunol2010 January 1; 184 (1): 327-35).In 4 grades of (BSL4) laboratorys of bio-safety of the US Army Medical Research Institute of Infectious Diseases of Maryland State Frederick Taylor, under the highest protection, process all ZEBOV infecting mouses.

1.10. antibody titer is measured

Anti-ZEBOV specific antibody is by aforementioned elisa assay (Hevey etc., Virology1998 November 10; 251 (1): 28-37).The serum of Mice Inoculated dilutes ELISA titration by the terminal on 96 orifice plates of the inactivation ZEBOV virion coating of purifying with sucrose, and dyes with the goat anti-mouse IgG Ab of peroxidase labelling.Titration is highly diluted, and it is non-specific or without hole (Warfield etc., the Vaccine2004 JIUYUE 3 days of antigen that wherein the absorbance of sample is greater than (twice); 22 (25-26): 3495-502).Anti-ZEBOV antibody titer is also used 96 orifice plate parallel assays of rVSV-ZEBOVGP coating.With the plate of VSV coating, as specificity, contrast.The titration onboard of the serum of Mice Inoculated, with the goat anti-mouse IgG antibody staining of peroxidase labelling, and antibody titer is measured as mentioned above.

For facs analysis, at the HEK293-ZEBOVGP cell alive of surface expression ZEBOV GP or with contrast HEK293 cell then anti-mouse antibodies dyeing of PE coupling (BD Biological Science Co., Ltd (BD Biosciences)) of serum through inoculation with 1 μ l of empty carrier transfection.Cell is analyzed in FACSCanto II flow cytometer (BD Biological Science Co., Ltd).

1.11.rVSV-ZEBOVGP plaque reduces experiment

Anti-ZEBOV neutralizing antibody reduces experiment by plaque and analyzes.The Mice Inoculated serum of five times of dilutions is with 100pfu rVSV-ZEBOVGP or contrast VSV mixing.Under 5% guinea pig serum fill-in exists, hatch sample 1h for 37 ℃, and carry out the experiment of standard plaque in the overlapping Vero E6 cell of the culture medium with comprising 1% Bacto-agar (Di Fuke company (DIFCO)).The percentage ratio that plaque reduces by relatively and the pfu number of sample calculate with input virus, Wilson etc. and Warfield etc., the same.

1.12. intracellular cytokine dyeing

Intracellular cytokine dyeing is undertaken by hatching Mice Inoculated splenocyte with EBOV GP peptide or ZEBOVGP-Fc, as is described in (Olinger etc., J Virol2005 November; 79 (22): 14189-96).Simply say, splenocyte by make spleen channel cross net filtration separated, washing and in the RPMI1640 culture medium that comprises 10%FBS, 2mM L-glutamine, 1mM HEPES and 0.1mM non essential amino acid, cultivate.Three kinds of EBOV GP specific peptides (LYDRLASTV, VSTGTGPGAGDFAFHK and EYLFEVDNL) of 1 μ g/ml for splenocyte (Olinger etc., the same and Warfield etc., J Immunol2005 July 15; 175 (2): 1184-91) or as the uncorrelated peptide (KINSTALL) of negative control stimulate.Or splenocyte stimulates with 10 μ g/ml ZEBOVGP-Fc or FC albumen.Stimulate and interleukin-22 exist under 37 ° of C hatch after 1h, add brefeldin A, and cell hatches 5h, then with the anti-mice CD8mAb clone RM4-5 (hero company) of APC coupling, dye.Then cell washs, fixing, and uses penetrating dose of (cytofix/cytoperm) test kit of fix/cell of cell (BD Biological Science Co., Ltd) thoroughly to change according to manufacturer's description.Rat anti-mouse IFN-γ mAb clone XMG1.2 (hero company) dyeing of PE coupling for IFN-γ in born of the same parents, and by the flow cytometry analysis in FACSCanto II equipment.

1.13. statistical analysis

Significance,statistical between two meansigma methodss is by not matching student t check mensuration, and use Graph Pad computed in software, and p-value is included in text and figure.

2. result

2.1.ZEBOVGP-Fc fusion rotein experience furin cutting, and the complicated processing of natural ZEBOV GP.

In order to express great amount of soluble ZEBOV GP for purification, qualitative and immune object, developed the stable Chinese hamster ovary celI system of expressing the ZEBOV GP ectodomain that is fused to human IgG1 Fc fragment.Inference Fc fragment can increase output, simplify purification, and maximizes in vitro and in vivo stability (Chamow etc., Trends Biotechnol1996 February of restructuring soluble protein; 14 (2): 52-60).Also comprise FLAG label epi-position between ZEBOV GP and Fc fragment with the expression with anti-FLAG M2mAb monitoring chimeric protein, and also can use enterokinase (at the restricted protease of FLAG label site cutting) to excise Fc fragment.For this reason, the encode plasmid of dihydrofolate reductase (DHFR) gene of pDHIP(for CHO dhfr-cell) and pEF-ZEBOVGP-Fc(coding be fused to the construct of the ZEBOV GP extracellular domain of IgG1Fc fragment) cotransfection (Figure 1A).The plasmid of the identical Fc fragment that in contrast, CHO dhfr-cell also comprises FLAG label with pDHIP and pEF-FLAG-Fc(coding N-end) cotransfection.Select single cell clone, and the overexpression of recombiant protein is realized (Silberstein etc., the same) by increasing MTX concentration.As ELISA, surveying the Chinese hamster ovary celI of the highest ZEBOVGP-Fc of generation or FLAG-Fc protein yield clones for generating albumen.The albumen of protein A purification is by the SDS-PAGE under reducing condition and Coomassie blue stain analysis (Figure 1B) subsequently.On ZEBOVGP-Fc, observe two main bands: have the wide band of approximately 130-150kDa of the high-glycosylation protein specificity of GP1 expection molecular weight, and have the little band of approximately 60kDa of GP2-Fc expection molecular weight.In ZEBOVGP-Fc swimming lane, also observe other little bands of corresponding part glycosylation or protein degradation.Contrast FLAG-Fc albumen moves into 36kDa band.With the Western engram analysis that anti-GP1mAb13F6-1-2, anti-FLAG mAb M2 and anti-Fc Ab survey, confirmed the characteristic (Fig. 1 C) of GP1, GP2-Fc and FLAG-Fc band.Data show ZEBOVGP-Fc fusion rotein, through the complicated post translational modification of post-mature GP, comprises the furin cutting between GP1 and GP2.

2.2.ZEBOVGP-Fc the ZEBOV GP in is assembled into homotrimer

The ZEBOVGP-Fc (50 μ g) by size exclusion chromatogram purification analyzes and has disclosed this fusion rotein and move into about 1 through Superdex20010/300GL post to use AKTA FPLC system (GE company), the main peak that has wide shoulder of 000kDa, this with as SDS-PAGE, analyze viewed GP high glycosylation characteristic consistent (Fig. 2 A).Because GP forms homotrimer in virus and cell surface, suppose that larger ZEBOVGP-Fc complex analysis is attributable to GP homotrimer and forms, and the ZEBOV-Fc of 6 copies can be connected to form by the disulfide bond of Fc fragment 2 homotrimers, its size is about 1,000-1,200kDa.In order to detect our hypothesis, with restricted enterokinase (40ng) digestion ZEBOVGP-Fc (100 μ g), the FLAG peptide between enterokinase cutting GP2 and Fc fragment.The size exclusion chromatograph of this digestion product has disclosed 3 main peaks, and approximately 600,140,50kDa (Fig. 2 A).Measured the carrier protein existing in the corresponding enterokinase goods in 140kDa peak.The GP homotrimer migration that described 600kDa peak forms with GP1 (130-150kDa) and GP2 (25kDa) is consistent.There is the big or small Fc fragment of expection (2 25kDa chains that disulfide bond connects) at described 50kDa peak, and moves altogether (data do not show) with Fc fragment under the same conditions.The Western engram analysis at 600 and the 50kDa peak of surveying with anti-GP1 and anti-Fc antibody has confirmed the corresponding GP ectodomain in described 600kDa peak, and the corresponding Fc fragment (Fig. 2 B) in described 50kDa peak.These results are clear has shown that the GP in ZEBOVGP-Fc has formed homotrimer, the natural GP form conformation of virus-like particle and cell surface expression (Hood etc., J Virol2010 March; 84 (6): 2972-82).

2.3.ZEBOVGP-Fc cause the anti-ZEBOV humoral response in Mice Inoculated

In order to measure ZEBOVGP-Fc, whether be immunogenicity, with ZEBOVGP-Fc or 8 C57BL/6 mices of contrast FLAG-Fc inoculation, and within 2 weeks, by ELISA, test anti-GP antibody after final reinforcement.The mice of described 8 inoculation ZEBOVGP-Fc develops 1:64,000-1:4, and 000 anti-ZEBOV GP antibody titer, as tested measured (Fig. 3 A) by ELISA with what kill in 96 orifice plates of radiation ZEBOV coating.Also use 96 orifice plates of the restructuring VSV coating of purification to carry out similar ELISA, described VSV comprises ZEBOV GP, rVSV-ZEBOVGP or wild type VSV contrasts as specificity.The acquisition in radiation ZEBOV ELISA of tiring with killing of measuring in described rVSV-ZEBOVGP ELISA is tired consistent.The serum of contrast FLAG-Fc Mice Inoculated with through radiation ZEBOV or rVSV-ZEBOVGP does not react, and has indicated the antibody detecting in inoculation animal special to ZEBOV GP.In addition, the serum of ZEBOVGP-Fc and FLAG-Fc inoculation animal does not react with wild type VSV, has indicated the antibody detecting in described ELISA to ZEBOV GP high special.

The serum of Mice Inoculated is also above tested by facs analysis by the HEK293 cell alive at cell surface expression EBOV GP albumen (HEK293-ZEBOVGP) or carrier transfectional cell (HEK293).The serum of ZEBOVGP-Fc immune mouse and HEK293-ZEBOVGP cell effect, but not with HEK293 cell effect (Fig. 3 B).Should be noted that an immune animal reacts poor (animal #8) with ZEBOV GP.As expected, the serum of FLAG-Fc-inoculation animal not with HEK293-ZEBOVGP or HEK293 cell effect, and anti-ZEBOV GP mAb13F6-1-2 only with HEK293-ZEBOVGP cell effect.These data and ELISA data consistent, and shown that the anti-ZEBOV GP antibody in ZEBOVGP-Fc inoculation animal reacts with natural ZEBOV GP.

2.4. the restructuring VSV that anti-ZEBOVGP antibody neutralization comprises ZEBOV GP.

Because institute's Mice Inoculated develops anti-ZEBOV GP antibody, can interesting these antibody of measuring neutralize virus.For this reason, the plaque of carrying out based on rVSV-ZEBOVGP neutralization reduces test, and rVSV-ZEBOVGP is the VSV that replication form restructuring VSV-G deletes, and contains ZEBOV GP by ZEBOV GP infection cell (Lawson etc., and Garbutt etc., the same).Because the neutralizing antibody for ZEBOV points to the only envelope glycoprotein in GP(virocyte surface), the neutralization of the neutralization simulation ZEBOV of rVSV-ZEBOVGP.What is interesting is, in the whole mices with ZEBOVGP-Fc inoculation, except one (the 8th), all develop neutralizing antibody (Fig. 4), and neutralization is antibody dilution dependency.The 8th mice serum also shown that low ELISA tires (Fig. 3 A) and to a little less than the combination of HEK293-ZEBOVGP cell, as facs analysis is evaluated (Fig. 3 B).In processing not with the mice serum of FLAG-Fc inoculation and rVSV-ZEBOVGP, and cause background to tire to decline 40% or lower, this has nothing to do with antibody dilution.These data show develop the neutralizing antibody to ZEBOV with whole mices (except the 8th) of ZEBOVGP-Fc inoculation.

2.5.ZEBOVGP-Fc induce the CD8 in Mice Inoculated ⁺t-cell effect

Because the protection of infecting for Filovirus mediates by body fluid and cellular immunization, analyzed with the T-cellular immunization in ZEBOVGP-Fc Mice Inoculated.

Use BALB/c mouse whether also can induce the immunity in different mouse species to measure ZEBOVGP-Fc.ZEBOVGP-Fc or FLAG-Fc inoculation for the group of 4 BALB/c mouse, used with regard to C57BL/6 mice identical scheme used.Described ZEBOVGP-Fc-inoculation BALB/c mouse also develops the anti-ZEBOV GP of the high-titer antibody (Fig. 5 A) of being evaluated as rVSV-ZEBOVGP virion ELISA, and rVSV-ZEBOVGP plaque reduces the neutralizing antibody (Fig. 5 B) of measuring.Final immunity, after 8 days, imposes euthanasia to mice, collects kidney, and separated nephrocyte, and stimulates with ZEBOVGP-Fc or ZEBOV GP peptide.Compare FLAG-Fc, in the splenocyte of the ZEBOVGP-Fc-Mice Inoculated stimulating with ZEBOVGP-Fc, observe IFN-γ-positive CD8 ⁺cell significantly increases (Fig. 6 A-C).In the splenocyte of the FLAG-Fc-Mice Inoculated stimulating with ZEBOVGP-Fc or FLAG-Fc, do not observe IFN-γ-positive CD8 ⁺cell increases.These data have indicated ZEBOVGP-Fc to induce anti-ZEBOV GP IFN-γ-positive CD8 ⁺the remarkable increase of cellular level.Should be noted that Fc fragment is IFN-γ-positive CD8 ⁺the weak derivant of cell, and cause the background level T cell activation from ZEBOVGP-Fc and FLAG-Fc Mice Inoculated.Unexpectedly, compare (Fig. 6 E) with FLAG-Fc-Mice Inoculated, ZEBOV GP specific peptide does not activate the IFN-γ-positive CD8 in ZEBOVGP-Fc-Mice Inoculated ⁺cell (Fig. 6 D).

2.6.ZEBOVGP-Fc protecting mice to resist ZEBOV attacks

Result shows that ZEBOVGP-Fc causes body fluid and the cell immune response in Mice Inoculated.Whether therefore, tested ZEBOVGP-Fc inoculation can protect mice to resist lethal ZEBOV attack.ZEBOVGP-Fc or FLAG-Fc inoculation for C57BL/6 mice, and with 1,000pfu mice adaptability ZEBOV, attack (Fig. 7) in 7 weeks after inoculation for the first time.With 7 in 8 mices of ZEBOVGP-Fc inoculation survivals in ZEBOV attacks, and accept in 8 mices of FLAG-Fc vaccine 7, in attack, die in latter 10 days.Data show EBOVGP-Fc causes the protective immunological reaction of attacking for ZEBOV in mice.

3. discuss

Result has herein shown that the GP extracellular domain of ZEBOVGP-Fc fusion rotein has experienced complicated post translational modification, comprises film in conjunction with furin cutting and the formation of the disulfide bond between GP1 and GP2 subunit of total length GP.In addition, the FPLC of the ZEBOVGP-Fc of enterokinase digestion analyzes and has shown that the ZEBOVGP fragment of Fc fusion rotein forms trimer, the similar natural GP at peplos and cell surface expression.ZEBOVGP-Fc inoculation causes that identification cell and virion surface film are in conjunction with the humoral immune reaction of GP.What is interesting is; anti-GP antibody and the ZEBOV of non-activity and the reacting phase of rVSV-ZEBOVGP are seemingly; described rVSV-ZEBOVGP is loaded with the replication form restructuring VSV granule of ZEBOV GP at virus surface; induce protective response (Feldmann etc., the PLoS Pathog2007 January of inhuman Primate (NHP); 3 (1): e2; Jones etc., Nat Med2005 July; 11 (7): 786-90).In anti-ZEBOVGP-Fc antibody and rVSV-ZEBOVGP, indicated this fusion rotein also to cause the protectiveness neutralizing antibody in NHP.These data show rVSV-ZEBOVGP be the instrument of putting into practice of evaluating overall and neutralizing antibody under BSL-2 condition, and indicated be loaded with the restructuring VSV of Filovirus GP can be for evaluating vaccine potential, evaluate the concordance of vaccine in generating, and as the surrogate markers thing of efficacy of vaccines in clinical trial.

Take and generate the cellular immunization that TNF-α and INF-γ are feature and in the protection of infecting for Filovirus, work (Hensley etc., PLoS Pathog2010 May; 6 (5): e1000904, and list of references wherein).T-cell effect in ZEBOVGP-Fc inducing mouse, IFN-γ in the splenocyte being stimulated by ZEBOVGP-Fc-positive CD8 ⁺the activation of cell proves.What is interesting is, the stimulation of FLAG-Fc causes background level reaction, has indicated Fc fragment minimum to the effect of the cell effect of fusion rotein.In NHP and people; human IgG1 Fc fragment in ZEBOVGP-Fc may be regarded self antigen as; and can by with antigen-presenting cell on Fc γ acceptor interaction immunogenicity (Zhang etc., Vaccine2009 February 5 of strengthening ZEBOVGP-Fc fusion rotein; 27 (6): 857-63; Guyre etc., the Cancer Immunol Immunother1997 11-12 month; 45 (3-4): 146-8; Chen etc., Retrovirology2007; 4:33).Aspect the unclear why GP-specific T-cells of described GP synthetic peptide in activation ZEBOVGP-Fc Mice Inoculated, there is no effect.In two researchs, these peptides have shown is testing the ZEBOV GP-specific T-cells that activates under condition of similarity used in mice (Olinger etc., and Warfield etc., the same) with us.Yet one of them peptide (LYDRLASTV) can not activate IFN-γ-positive CD8 in the BALB/c mouse with replication defect type Ebola Δ VP30 virus inoculation ⁺cell (Halfmann etc., J Virol2009 April; 83 (8): 3810).Whether need other to study to measure ZEBOVGP-Fc inoculation causes the not cell effect of T-cell epitope on the same group.In a word, clear cell and the humoral immunization of ZEBOVGP-Fc induction to ZEBOV GP extracellular domain that shown of these results.

A mice not survival in the lethal attack of ZEBOV with ZEBOVGP-Fc inoculation.Unfortunately, in attack, do not use Individual identification thing, and serology and survival data clearly can not be associated.Yet, the ZEBOVGP-Fc inoculation animal that there is no survival in attack may be develop can not in and only mice in the low-level anti-ZEBOV GP antibody group of rVSV-ZEBOVGP.Whether project measures level for the neutralizing antibody of replication form restructuring rVSV-ZEBOVGP to relevant for the protection of the lethal attack of ZEBOV.

These data indications ZEBOVGP-Fc can be as the subunit Filovirus vaccine of people's application.Previously about exploitation, the trial of the Filovirus subunit vaccine based on expressed soluble form Filovirus GP in insect cell provided in guinea pig model the limited protection of lethal attack (Mellquist-Riemenschneider etc., Virus Res2003 April; 92 (2): 187-9; Hevey etc., Virology1997 December 8 days; 239 (1): 206-16).Glycosylation in insect cell may have the stability of affected epi-position structure, homotrimer formation and soluble g P albumen, and these are the factors that possible cause soluble g P vaccine poor performance.The ZEBOVGP-Fc using in this research generates in mammalian cell, and the similar natural GP of most probable, is better than the soluble g P of expressed in insect cells.In addition; Fc label in ZEBOVGP-Fc is given our several advantages of vaccine scheme; comprise use temperate condition by the simplification of protein A column purification, the protein stability that Fc fragment produces increases and with antigen-presenting cell on Fc γ the acceptor interaction adjuvant effect (Zhang etc., the Guyre etc. that cause; Chen etc., the same).

These results have shown that inoculation ZEBOVGP-Fc fusion rotein causes the high level protection that ZEBOV is attacked, and have pointed out the subunit vaccine based on Filovirus GP-Fc fusion rotein can develop the protection for viral infection.This Filovirus GP-Fc can as independent vaccine or for example the current DNA vaccination of developing, virion and vector-viral vaccine be combined with other strategies.Subunit production of vaccine based on Filovirus GP-Fc fusion rotein is simple, easily purification, meet cost benefit, and the side effect causing is limited.

Embodiment 2

With the construct that comprises the whole ectodomains of Zaire Ebola virus glycoprotein (ZEBOVGP) that merge human IgG1's Fc fragment (ZEBOVGP-Fc), in Cavia porcellus and macaque, test the immunogenicity of FiloGP-Fc.Animal develops the Zaire Ebola pseudotype virus of strong anti-ZEBOVGP antibody response and serum neutralization.Albumen generation, purification, the method for measuring anti-ZEBOVGP antibody and virus neutralization tests by ELISA are described in embodiment 1.Herein data show FiloGP-Fc albumen in Cavia porcellus and macaque, have hyperimmunization originality, and cause at Filovirus and infect the neutralizing antibody of finding in the convalescence patients serum of survival.

Immunogenicity in Cavia porcellus

Cavia porcellus strain 13 use are dissolved in the purified ZEBOVGP-Fc of 0.1mg or the inoculation of Fc fragment of QS-21 adjuvant.Animal is strengthened 2 times with 3 weekly intervals with the corresponding vaccine of same dose.Latter 2 weeks of final reinforcement, Cavia porcellus blood-letting, and the serum of collecting is for antibody analysis.All the serum of ZEBOVGP-Fc inoculation animal comprises the high antibody titer for ZEBOVGP, and as virion, ELISA evaluates (Fig. 8), and scope is 1/32,000-1/128, and 000.Contrast Cavia porcellus does not only comprise anti-ZEBOVGP antibody with serum before the inoculation of Fc fragment inoculation (Fig. 9) and all animals, has indicated anti-ZEBOVGP antibody response special to viral glycoprotein.

For analyze anti-Ebola virus antibody that ZEBOVGP-Fc vaccine causes whether can in and Ebola virus, together with the false type of serum (1:25 dilution) and ZEBOVGP of inoculation animal, hatch, and on 96 orifice plates that comprise Vero E6 cell monolayer, by terminal, dilute neutralization test and evaluate to neutralize and tire.After 48h is hatched, the cytopathic effect causing with regard to viral infection is manhole under the microscope.Compare with the virus that preimmune serum is processed, calculate percent neutralization (Figure 10).This neutralization test has clearly shown that ZEBOVGP-Fc inoculation has caused anti-Ebola virus neutralizing antibody, and only Fc fragment can not cause neutralizing antibody.

Immunogenicity in macaque

Because the result in mice and Cavia porcellus is clear, shown that ZEBOVGP-Fc has hyperimmunization originality, and the anti-Ebola virus antibody of induction neutralization, test described recombiant protein and whether also in macaque (Maccacamulata), cause immunoreation to measure it.For this reason, first measure serum of macaque and whether comprise anti-Ebola virus antibody.The serum that virion ELISA has disclosed three monkeys of using in research does not comprise anti-Ebola virus antibody (Figure 11).

2 monkeys of ZEBOVGP-Fc inoculation of then purifying with 0.4mg, and only by 0.4mg Fc fragment, use poly-(IC) as 1 contrast monkey of adjuvant inoculation.After primary vaccination 3 weeks, described monkey blood-letting, and then with the corresponding vaccine of same dose, strengthen.Strengthen latter three weeks, monkey blood-letting, and blood serum sample is for antibody analysis.The serum of ZEBOVGP-Fc inoculation animal comprise approximately 1/32,000 for the high antibody titer of ZEBOVGP, as virion, ELISA evaluates (Figure 12).As expected, only with the contrast monkey of Fc fragment inoculation, do not develop anti-ZEBOVGP antibody, indicated described anti-ZEBOVGP antibody response special to viral glycoprotein.

In order to measure the monkey of ZEBOVGP-Fc vaccination, whether develop anti-Ebola virus neutralizing antibody, the serum (1:50 dilution) of inoculation animal is hatched together with the false type of ZEBOVGP, and on 96 orifice plates that comprise Vero E6 cell monolayer, by terminal, dilutes neutralization test evaluation neutralization and tire.After 48h is hatched, the cytopathic effect causing with regard to viral infection checks 96 orifice plates under the microscope.Compare with the virus that preimmune serum is processed, calculate percent neutralization (Figure 13).

Sum up

Herein data are clear has shown that described ZEBOVGP-Fc vaccine induces the neutralization of high resistance Ebola virus to tire in Cavia porcellus and monkey.In addition, this vaccine causes to protect and resists the anti-Filovirus neutralizing antibody that Filovirus infects.

Should be understood that embodiment as herein described and embodiment are only for illustration purpose, it will be understood by a person skilled in the art that various modifications or the change made accordingly, and they are included in the application's spirit and the scope of authority and appended claims.That quotes herein allly delivers thing, patent and patent application and includes in by reference of text herein for all objects.

Claims (35)

1. a fusion rotein that comprises Filovirus glycoprotein fragment and immunoglobulin polypeptides fragment.

2. fusion rotein as claimed in claim 1, is characterized in that, described Filovirus glycoprotein fragment is ectodomain.

3. fusion rotein as claimed in claim 1, is characterized in that, described Filovirus glycoprotein fragment is from Ebola virus.

4. fusion rotein as claimed in claim 3, is characterized in that, described Ebola virus is Zaire Ebola virus Mayinga strain.

5. fusion rotein as claimed in claim 1, is characterized in that, described immunoglobulin polypeptides fragment is immunoglobulin heavy chain constant region polypeptide.

6. fusion rotein as claimed in claim 1, is characterized in that, described immunoglobulin is IgG1.

7. fusion rotein as claimed in claim 1, is characterized in that, described fusion rotein also comprises the joint between Filovirus glycoprotein fragment and immunoglobulin polypeptides fragment.

8. fusion rotein as claimed in claim 1, is characterized in that, described fusion rotein is just like the sequence shown in SEQ ID NO:2.

9. an immunogenic composition that comprises fusion rotein as claimed in claim 1.

10. immunogenic composition as claimed in claim 9, is characterized in that, described compositions also comprises adjuvant.

11. a nucleic acid carrier, described carrier comprises the nucleic acid sequence encoding of the fusion rotein that contains Filovirus glycoprotein fragment and immunoglobulin polypeptides fragment.

12. nucleic acid carriers as claimed in claim 11, is characterized in that, described Filovirus glycoprotein fragment is ectodomain.

13. nucleic acid carriers as claimed in claim 11, is characterized in that, described Filovirus glycoprotein fragment is from Ebola virus.

14. nucleic acid carriers as claimed in claim 13, is characterized in that, described Ebola virus is Zaire Ebola virus Mayinga strain.

15. nucleic acid carriers as claimed in claim 11, is characterized in that, described immunoglobulin polypeptides fragment is immunoglobulin heavy chain constant region polypeptide.

16. nucleic acid carriers as claimed in claim 11, is characterized in that, described immunoglobulin is IgG1.

17. nucleic acid carrier as claimed in claim 11, is characterized in that, described nucleic acid carrier also comprises the nucleic acid sequence encoding of joint between Filovirus glycoprotein fragment and immunoglobulin polypeptides fragment.

18. nucleic acid carriers as claimed in claim 11, is characterized in that, the nucleic acid sequence encoding of described fusion rotein has the sequence shown in SEQ ID NO:1.

Induce the immunoreactive method of protection in patient, Filovirus being infected for 19. 1 kinds, described method comprises the immunogenic composition as claimed in claim 9 that gives patient's immunity effective dose.

20. methods as claimed in claim 19, is characterized in that, described immunogenic composition intramuscular gives.

21. method as claimed in claim 19, is characterized in that, described method also comprises the step of the immunogenic composition that contains the second Filovirus antigen.

22. methods as claimed in claim 21, is characterized in that, described the second antigen is expressed by recombinant viral vector.

Induce the immunoreactive method of protection in patient, Filovirus being infected for 23. 1 kinds, described method comprises the nucleic acid carrier as claimed in claim 11 that gives patient's immunity effective dose.

24. in detection patient, to the immunoreactive method of Filovirus, described method comprises makes patient's biological sample contact fusion rotein as claimed in claim 1, and detects immunoreation.

25. method as claimed in claim 24, is characterized in that, the step of antibody and fusion rotein combination in the immunoreactive step inclusion test of described detection biological sample.

26. methods as claimed in claim 25, is characterized in that, the step of described detection antibodies is undertaken by ELISA, chemoluminescence test or fluorescent test.

27. methods as claimed in claim 24, is characterized in that, the step of the immunoreactive step inclusion test of described detection cell immune response.

28. method as claimed in claim 27, is characterized in that, the step of described detection cell immune response is undertaken by detecting IFN-γ or TNF-α.

29. 1 kinds of methods that detect neutralizing antibody in patient's biological sample, described method comprises makes biological sample contact be subject to the Filovirus cell infecting and the recombinant vesicular stomatitis virus (VSV) of expressing Filovirus GP.

30. methods as claimed in claim 29, is characterized in that, described restructuring VSV is express fluorescent protein also.

31. methods as claimed in claim 29, is characterized in that, described restructuring VSV expresses Ebola and expresses GP.

32. methods as claimed in claim 29, is characterized in that, described Ebola virus is Zaire Ebola virus Mayinga strain.

33. methods as claimed in claim 29, is characterized in that, described in to be subject to the cell that Filovirus infects be Vero E6 cell.

34. 1 kinds of methods that detect anti-Filovirus antibody in patient's biological sample, described method comprises makes biological sample contact be combined in the restructuring VSV on solid support, and detects the anti-Filovirus antibody in conjunction with restructuring VSV.

35. methods as claimed in claim 34, is characterized in that, described anti-Filovirus antibody is used ELISA, chemoluminescence method or Fluorometric assay.

Images